T-SPOT.TB Reactivity in Southern African Children With and Without in Utero Human Immunodeficiency Virus Exposure

Abstract Infants who are human immunodeficiency virus (HIV)-exposed uninfected (iHEU) experience higher risk of infectious morbidity than infants HIV-unexposed uninfected (iHUU). We compared tuberculosis (TB) infection prevalence in 418 Bacillus Calmette-Guérin vaccinated sub-Saharan African iHEU and iHUU aged 9–18 months using T-SPOT.TB. Prevalence of TB infection was low and did not differ by HIV exposure status.

Children under 5 years of age experience high risk of progression to tuberculosis (TB) disease if untreated [1]. In sub-Saharan Africa, about 25% of persons of childbearing potential are diagnosed with human immunodeficiency virus (HIV) [2]. Successful antiretroviral treatment (ART) scale-up for pregnant persons with HIV has resulted in an increasing number of infants born HIV-exposed uninfected (iHEU).The iHEU experience greater risk of infectious morbidity than infants born HIV-unexposed uninfected (iHUU) [3].
Bacillus Calmette-Guérin (BCG) vaccine prevents severe TB disease. BCG vaccination induces T-cell interferon-gamma (IFN-γ) production, an important component of protection against TB [4]. We previously found a significantly lower proportion of BCG-specific IFN-γ producing CD4 + T cells among iHEU, suggesting that iHEU may not achieve equivalent BCG immune protection compared to iHUU [5]. Therefore, we investigated TB infection prevalence by HIV exposure status among BCG-vaccinated infants in Botswana and South Africa (SA), 2 high burden HIV and TB settings.

Study Design
The study was nested within 2 prospective observational cohort studies enrolling pregnant women with and without HIV and their infants. The Tshilo Dikotla study and the Innate Factors Associated with Nursing Transmission (InFANT) study recruited participants from government antenatal clinics in Botswana and SA between 2013 and 2020 [6,7]. Infants with severe birth complications, or born to mothers with active TB or TB symptoms were excluded. All infants received BCG vaccination within 72 hours of birth. Participants were followed over 36 months in Botswana and 12 months in SA. Peripheral blood mononuclear cells were collected at 9-12 and 18 months in Botswana, at 9 and 12 months in SA, and stored in liquid nitrogen.

Ethics
This study was approved by the Health Research Development Committee in Botswana, Massachusetts General Hospital's Institutional Review Board, and University of Cape Town's Human Research Ethics Committee. Women provided written informed consent for their participation and that of their infant.

T-SPOT.TB Assay
T-SPOT.TB assays (Oxford Immunotec) were performed and interpreted according to manufacturer's instructions. Samples below the recommended cell number were normalized as previously described [8]. For invalid or borderline results, retesting was performed using an aliquot collected at the same visit or a follow-up visit. If the re-tested result was valid, the valid result was assigned to the initial visit. Infants testing BRIEF REPORT • CID 2023:77 (15 October) • 1133

Clinical Infectious Diseases
T-SPOT.TB positive were referred to government clinics. We defined "TB infection" as T-SPOT.TB positive without TB disease symptoms at the time of specimen draw.

Sensitivity Analysis
Due to timing variation of testing between sites, we simulated results as if all testing was performed at 12 months. For SA infants, we assumed a positive test at month 9 would have a negative at month 12 with probability R (reversion), and infants with a negative or invalid result at month 9 would have a positive at month 12 with probability C (conversion). These probabilities were applied to 12-to 18-months results in the Botswana cohort. We assumed a baseline P (prevalence) at 12 months to calculate the probability of a positive (or negative/invalid) test at 18 months having been negative (or positive) at month 12. For each combination of R, C, and P, ranging from 0% to 20% based on published studies [8,9], we randomly generated 5000 data sets and performed Fisher exact tests on the pooled month 12 data, comparing the proportion of positive results by infant HIV exposure status.

Statistical Analysis
Data analysis was performed using R (version 4.0.4). Normally distributed continuous variables were compared by t test using means with standard deviations. Continuous variables with skewed distributions were compared by Wilcoxon rank-sum test using medians with interquartile ranges. Categorical variables were compared by χ 2 test. TB infection proportions were compared by infant HIV exposure status using Fisher exact test.

Power Calculations
Previous sub-Saharan data reported a prevalence of TB infection of 10.9% (95% confidence interval [CI], 6.1%-17.7%) in 6-month-old iHEU [8]. Thus, we expected that at least 18% of iHEU would test positive by 12 months. Given our study's sample size (125 iHUU and 293 iHEU), we had at least 80% power to detect a 57.5% difference in TB infection at 12 months, assuming a prevalence of ≤ 7.65% among iHUU.

Cohort Characteristics
The study included 418 mother-infant pairs, of which 293 were iHEU (Supplementary Table 1). The proportion of iHEU was higher in Botswana compared to SA. Infant sex and gestational age at birth were similar between HIV exposure groups. Women with HIV were older and had higher gravidity than women without HIV. Among women with HIV, 63.0% were on ART at conception with median CD4 count of 463 cells/ mm 3 at enrollment. Fifteen (3.6%) infants had household TB exposure cases (n = 5 in Botswana; n = 10 in SA), including 6 mothers of infants in SA. Household exposure did not differ by HIV exposure status.

or by study site (Supplementary
We conducted a sensitivity analysis to impute T-SPOT.TB results at 12 months of age across study sites, considering potential conversion and reversion rates over time (Supplementary  Table 3). No combination of assumptions gave a statistically significant difference between HIV-exposure groups in T-SPOT.TB positivity more than 5% of the time.

DISCUSSION
In our Southern African cohort, we found a low overall risk of TB infection among infants BCG-vaccinated at birth (3.3%), without variation by HIV exposure status. The lack of difference is important, as iHEU have been reported to be at high risk of infectious morbidity [3]. Although testing was performed between 9 and 18 months of life, with some infants having a longer window of risk for TB exposure, the sensitivity analysis suggests that the prevalence of T-SPOT.TB positivity was robust to conversion and reversion between the observed Reason of invalid: contamination of kits or assay (n = 2); PHA < 20 SFCs (n = 7) and Nil control > 10 SFCs (n = 6).
time points. Literature investigating TB infection in iHEU using interferon-gamma release assays (IGRAs) like the QuantiFERON-TB or T-SPOT.TB is limited [10], and studies including iHUU as a comparison group are scarce. To our knowledge, this is the largest study comparing TB infection prevalence between Southern African iHEU and iHUU using T-SPOT.TB. The prevalence of TB infection was lower among infants in this study compared to other studies using IGRA-based approaches [9]. Differences in cohort characteristics likely account for lower IGRA positivity in our study. We excluded mothers with active TB disease. Furthermore, the previously published SA study assessed TB infection in children with a mean age of 3.5 years [9], evaluating a longer exposure window. It was also conducted during a period when SA recorded its highest TB incidence in the last 2 decades [11]. Thus, household TB contact was more common than our study (13.2% vs 3.6%).
We found no difference in T-SPOT.TB reactivity by HIV exposure status. This differs from a Ugandan study where children HEU up to 5 years of age had higher IGRA positivity prevalence than children HUU [10]. Differences in maternal inclusion criteria and longer follow-up period likely explain the higher prevalence reported in Ugandan children who were HEU.
The strengths of this study include a large sample size, with cohorts recruited in neighboring countries, both with high HIV and TB burden, using a common protocol. Pooling of data increased study power. Although the timing of testing varied between sites, we employed a sensitivity analysis to assess for robustness of findings. Given the lower than anticipated T-SPOT.TB positivity prevalence, we did not have sufficient power to conclusively evaluate the association between HIV exposure and TB infection. Because the prevalence of a T-SPOT.TB reactivity in iHEU was 3.4% in our cohort, prevalence among iHUU would had to have been ≤0.175%, a 94.9% reduction, to detect a significant difference. Adequately powered studies would be needed to definitively exclude a higher risk of TB infection in iHEU.
We employed IGRA-based testing, similar to previous studies [9,10]. Infant T cells have lower IFN-γ producing capacity than adult T cells [12], and perinatal HIV exposure has been associated with altered immunity [13]. Thus, it is unclear whether iHEU and iHUU have similar T-SPOT.TB reactivity. Furthermore, IGRA testing is not recommended for children under 2 years of age, but tuberculin skin testing can result in false positive tests in BCG-vaccinated individuals. Assays targeting non-IFN-γ markers have been proposed as alternatives in BCG-vaccinated children under 2 years of age [14] and may be beneficial to use in future studies in parallel with IGRA testing.
In summary, we showed that the TB infection prevalence among BCG-vaccinated infants from 2 Southern African countries with high HIV and TB prevalence was low and did not vary by fetal HIV exposure status.

Supplementary Data
Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.